The invention relates to the field of microscopy, and particularly relates to the field of coherent anti-stokes Raman scattering microscopy.
Coherent anti-stokes Raman scattering (CARS) microscopy provides for the imaging of chemical and biological samples by using molecular vibrations as a contrast mechanism. In particular, CARS microscopy uses at least two laser fields, a pump electromagnetic field with a center frequency at xcfx89p and a Stokes electromagnetic field with a center frequency at xcfx89s. The pump and Stokes fields interact with a sample and generate a coherent anti-Stokes field having a frequency of xcfx89AS=2xcfx89pxe2x88x92xcfx89s in the phase matched direction. When the Raman shift of xcfx89pxe2x88x92xcfx89s is tuned to be resonant at a given vibrational mode, an enhanced CARS signal is observed at the anti-Stokes frequency xcfx89AS.
Unlike fluorescence microscopy, CARS microscopy does not require the use of fluorophores (which may undergo photobleaching), since the imaging relies on vibrational contrast of biological and chemical materials. Further, the coherent nature of CARS microscopy offers significantly higher sensitivity than spontaneous Raman microscopy. This permits the use of lower average excitation powers (which is tolerable for biological samples). The fact that xcfx89AS greater than xcfx89p, xcfx89s allows the signal to be detected in the presence of background fluorescence.
For example, U.S. Pat. No. 4,405,237 discloses a coherent anti-Stokes Raman spectroscopic imaging device in which two laser pulse trains of different wavelengths, temporally and spatially overlapped, are used to simultaneously illuminate a sample. The signal beam in the phase matching direction with a two-dimensional detector, which gives the spatial resolution.
U.S. Pat. No. 6,108,081 discloses a different method and apparatus for microscopic vibrational imaging using coherent anti-Stokes Raman scattering. In the apparatus of the ""081 patent, collinear pump and Stokes beams were focused by a high numerical aperture (NA) objective lens. The nonlinear dependence of the signal on the excitation intensity ensures a small probe volume of the foci, allowing three-dimensional sectioning across a thick sample. The signal beam is detected in the forward direction.
A prior art CARS imaging system (based on the ""081 patent) 10 is shown diagrammatically in FIG. 1, in which collinear pump and Stokes beams 12 at frequencies of xcfx89p and xcfx89s respectively, are directed to a microscope objective lens 16, and onto a sample 18. The CARS signal is detected in the forward direction, and is received by collecting optics 20, filtered by one or more filters 22, and detected by a detector 26.
The signal beam that is created in CARS imaging, however, includes a substantial amount of background with no vibrational contrast from which the signal must be filtered or somehow distinguished. For example, as shown in FIG. 2, a conventional (forward-detected) lateral CARS intensity profile of a 535 nm polystyrene bead embedded in water includes a substantial amount of CARS background from water 30 in addition to the characteristic CARS signal from the bead 32. The horizontal axis in FIG. 2 represents the lateral dimension (in xcexcm) across the scan area, and the vertical axis represents the strength of the CARS signal (in cts). The presence of this background from the isotropic bulk water has hindered efforts to increase the sensitivity of CARS imaging, particularly in biological applications. The CARS background is caused by a variety of circumstances. For example, because of electronic contributions to the third order nonlinear susceptibility, there exists a non-resonant contribution to the CARS signal of the sample of interest as well as of the surrounding isotropic bulk medium (i.e., solvent), which is independent of the Raman shift, surrounding isotropic bulk medium (i.e., solvent), which is independent of the Raman shift, xcfx89pxe2x88x92xcfx89S. In addition, in biological applications the common solvent water has strong resonant signals with broad spectral widths that may overwhelm the weak signal of the sample.
As shown in FIG. 3, a combined CARS image 40 and intensity profile 42 taken along line 44xe2x80x9444 of epithelial cells shows that the signal includes CARS background (as generally indicated at 46) that may not be easily distinguished from the microscopic sample signal (as generally indicated at 48). The lateral dimension (in xcexcm) is shown along the horizontal axis, and signal strength (in cts) is shown along the vertical axis. In certain embodiments, these bulk solvent background contributions to the detected CARS signal may overwhelm the CARS sample signals.
There is a need, therefore, for a system and method for providing improved sensitivity of CARS microscopy, and in particular, to provide a CARS system that reduces the background from the bulk medium, and hence provides a higher signal-to-background ratio.
The invention provides systems and methods for detecting a coherent anti-Stokes Raman scattering signal from a microscopic sample. In one embodiment, the system includes at least two laser sources, a pump source for generating an electromagnetic field at the pump frequency, a Stokes source for generating an electromagnetic field at the Stokes frequency that is different from the pump frequency, optics to direct collinearly the pump and Stokes beams toward an objective lens, which provides a common focal spot, and a detector for measuring a coherent anti-Stokes signal in the backward (epi) direction that is generated by the interaction of pump and Stokes fields with the sample, and collected by the same lens focusing the pump and Stokes beams.